The immediate purpose of the proposed research is to provide a high resolution x-ray structure determination for the enzyme dihydrofolate reductase from an amethopterin (methotrexate) resistant strain of Eschericia coli. Inhibitor binding in the active site and to the adjacent hydrophobic region will be examined with the expectation of making significant new contributions in two broad areas: the mechanism of action of dihydrofolate reductase and the rational development of drugs of chemotherapeutic importance. Large crystals of an E. coli dihydrofolate reductase-methotrexate complex have undergone preliminary x-ray diffraction characterization (space group P6 122, a triple bond b equals 93.71 A, c equals 74.09 A, one molecule per asymmetric unit) and have been shown to be suitable for high resolution x-ray studies. Crystals of the same enzyme from a mutant Lactobacillus casei organism have also been grown and the two enzyme structures will be solved in parallel. Experience gained in a comparative structural examination of the enzyme from two closely related species will hopefully suggest possible fruitful approaches for the rational design of new anticancer drugs which may attain a greater level of tissue specificity than is now possible with folate analogs such as methotrexate. BIBLIOGRAPHIC REFERENCES: Crystallographic Structure Refinement of Chromatium High Potential Iron Protein at Two Angstroms Resolution, S. T. Freer, R. A. Alden, C. W. Carter and J. Kraut, J. Biol, Chem. 250, 46 (1975). X-ray Crystallographic Study of Boronic Acid Adducts with Subtilisin BPN' (Novo): A Model for the Catalytic Transition State, D. A. Matthews, R. A. Alden, J. J. Birktoft, S. T. Freer and J. Kraut, J. Biol. Chem. 250, 7120 (1975).